The following abbreviations are used throughout this application:
A. cal.--Autographa californica PA0 AcMNPV--Autographa californica nuclear polyhedrosis virus PA0 bp--base pairs PA0 ECV--extracellular virus PA0 GV--granulosis virus PA0 kD--kilodaltons PA0 MOI--multiplicity of infection PA0 NPV--nuclear polyhedrosis virus PA0 OB--occlusion body PA0 OV--occluded virus PA0 PCR--polymerase chain reaction PA0 PDV--polyhedron derived virus PA0 p.i.--post-infection PA0 PIB--polyhedron inclusion body (also known as OB) PA0 5' UTR--the mRNA or gene sequence corresponding to the region extending from the start site of gene transcription to the last base or basepair that precedes the initiation codon for protein synthesis PA0 3' UTR--the mRNA or gene sequence corresponding to the region extending from the first base or basepair after the termination codon for protein synthesis to the last gene-encoded base at the 3' terminus of the mRNA PA0 (+)-strand--the DNA strand of a gene and its flanking sequences which has the same sense as the RNA that is derived from that gene PA0 (-)-strand--the DNA strand of a gene and its flanking sequences that is complementary to the (+) strand
The present invention applies to all insect viruses, including DNA and RNA viruses. The DNA viruses include entomopox viruses (EPV), and Baculoviridae viruses, such as nuclear polyhedrosis viruses (NPV) and granulosis viruses (GV), and the like. The RNA viruses include togaviruses, flaviviruses, picornaviruses, cytoplasmic polyhedrosis viruses (CPV), and the like. The Subfamily of double stranded DNA viruses Eubaculovirinae includes two genera, NPVs and GVs, which are particularly useful for biological control because they produce occlusion bodies (OBs) in their life cycle.
Over 400 baculovirus isolates have been described. The Autographa californica nuclear polyhedrosis virus (AcMNPV) is the prototype virus of the Family Baculoviridae and has a wide host range. The AcMNPV virus was originally isolated from Autographa californica (A. cal.), a lepidopteran noctuid (which in its adult stage is a nocturnal moth), commonly known as the alfalfa looper. This virus infects 12 Families and more than 30 species within the order of Lepidopteran insects (Bibliography entry 1). It is not known to infect productively any species outside this order.
The use of baculoviruses as bioinsecticides holds great promise. One of the major impediments to their widespread use in agriculture is the time lag between initial infection of the insect and its death. This lag can range from a few days to several weeks. During this lag, the insect continues to feed, causing further damage to the plant. A number of researchers have attempted to overcome this drawback by inserting a heterologous gene into the viral genome, so as to express an insect controlling or modifying substance, such as a toxin (2,3,4), neuropeptide and hormone (5,6) or enzyme (7).
While genetic engineering provides a means for overcoming technical obstacles to the commercialization of viruses as bioinsecticides, it has also given rise to a perception that there may be a second potential impediment to their widespread acceptance. In particular, there is speculation that release of these genetically engineered viruses into the environment may result in unforseen consequences to the ecosystem as these viruses replicate and spread (8,9). To date, all recombinant insect viruses produced for the biocontrol of insects would be subject to this speculation because all are capable of insect to insect transmission. Thus, there is a need for recombinant insect-specific viruses which have a reduced capacity for insect to insect (host-to-host) transmission following their release into the environment.
The life cycle of baculoviruses, as exemplified by AcMNPV, includes two stages. Each stage of the life cycle is represented by a specific form of the virus: Extracellular viral particles (ECV) which are nonoccluded, and occluded virus particles (OV) (10,11). The extracellular and occluded virus forms have the same genome, but exhibit different biological properties. The maturation of each of the two forms of the virus is directed by separate sets of viral genes, some of which are unique to each form.
In its naturally occurring insect infectious form, multiple virions are found embedded in a paracrystalline protein matrix known as an occlusion body (OB), which is also referred to as a polyhedron inclusion body (PIB). The proteinaceous viral occlusions are referred to as polyhedra (polyhedron is the singular term). A polyhedrin protein, which has a molecular weight of 29 kD, is the major viral-encoded structural protein of the viral occlusions (10,12). (Similarly, GVs produce OBs which are composed primarily of granulin, rather than polyhedrin).
The viral occlusions are an important part of the natural baculovirus life cycle, providing the means for horizontal (insect to insect) transmission among susceptible insect species. In the environment, a susceptible insect (usually in the larval stage) ingests the viral occlusions from a contaminated food source, such as a plant. The crystalline occlusions dissociate in the gut of the susceptible insects to release the infectious viral particles. These polyhedron derived viruses (PDV) invade and replicate in the cells of the midgut tissue (10).
It is believed that virus particles enter the cell by endocytosis or fusion, and the viral DNA is uncoated at the nuclear pore or in the nucleus. Viral DNA replication is detected within six hours. By 10-12 hours post-infection (p.i.), secondary infection spreads to other insect tissues by the budding of the extracellular virus (ECV) from the surface of the cell. The ECV form of the virus is responsible for cell to cell spread of the virus within an individual infected insect, as well as transmitting infection in cell culture.
Late in the infection cycle (12 hours p.i.), polyhedrin protein can be detected in infected cells. It is not until 18-24 hours p.i. that the polyhedrin protein assembles in the nucleus of the infected cell and virus particles become embedded in the proteinaceous occlusions. Viral occlusions accumulate to large numbers over 4-5 days as cells lyse. These polyhedra have no active role in the spread of infection in the larva. ECVs in the haemolymph multiply and spread, leading to the death of the larva (10,11,12).
When infected larvae die, millions of polyhedra remain in the decomposing tissue, while the ECVs are degraded. When other larvae are exposed to the polyhedra, for example, by ingestion of contaminated plants or other food material, the cycle is repeated (10).
In summary, the occluded form of the virus is responsible for the initial infection of the insect through the gut, as well as the environmental stability of the virus. PDVs are essentially not infectious when administered by injection, but are highly infectious orally. The non-occluded form of the virus (i.e., ECV) is responsible for secondary and cell to cell infection. ECVs are highly infectious for cells in culture or internal insect tissues by injection, but essentially not infectious by oral administration.
The use of recombinant baculoviruses expressing foreign proteins which are toxic to insects is facilitated by the fact that these viruses are not pathogenic to vertebrates or plants. In addition, the baculoviruses generally have a narrow host range. Many strains are limited to one or a few insect species.
The most widely studied baculovirus is AcMNPV. AcMNPV is known to infect 12 Families and more than 30 species within the insect order Lepidoptera (1). It is not known to infect productively any species outside this order. Both the general public and various regulatory agencies have discussed the potential consequences of a release of a strain of AcMNPV containing a foreign DNA sequence (8,9). This relates to the possibility that the engineered virus could spread into non-target lepidopterans. Another factor to consider is the known environmental stability of these viruses.
Thus, there is a need for recombinant insect-specific viruses with a reduced ability to spread from insect to insect (host to host). These viruses should be able to infect an insect, but should have a significant decrease in the ability to be transmitted from insect to insect, thereby limiting the persistence of the virus in the environment.